论文部分内容阅读
目的:克隆人乳头状瘤病毒(HPV)11型E2基因片段,构建真核生物表达载体,研究低危型HPV E2基因的表达对正常鼻咽部黏膜细胞(NP69-SV40T)增殖的影响作用。方法:提取HPV11型DNA,设计E2基因引物,PCR扩增E2基因全长序列,分子克隆至含有绿色荧光蛋白的pEGFP-C1表达载体,构建真核表达载体pEGFP-C1-E2,转染至NP69-SV40T细胞内,转染后行Western blot、流式细胞仪检测以及CCK8检测,观察对NP69-SV40T细胞增殖活性的影响。结果:成功克隆和构建了pEGFP-C1-E2表达载体,转入NP69-SV40T细胞后,证明了其在NP69-SV40T细胞的表达,CCK8和流式细胞仪检测细胞周期,实验结果表明低危型HPV E2转染NP69-SV40T细胞可以促进细胞增殖,同时Western blot检测细胞周期素CDK2和CDK4发现,在3组(转染组、对照组和空白组)中表达量无统计学差异。结论:表达E2的pEGFP-C1-E2真核表达质粒能有效转染NP69-SV40T,并且转染后E2基因的表达能促进细胞的增殖。这些结果为深入研究复发性呼吸道乳头状瘤病的发生和发展提供了新的思路和方法。
OBJECTIVE: To clone human papillomavirus (HPV) 11 E2 gene and construct eukaryotic expression vector to study the effect of low-risk HPV E2 gene on the proliferation of normal nasopharyngeal mucosal cells (NP69-SV40T). Methods: HPV11 DNA was extracted and E2 gene primers were designed. The full-length sequence of E2 gene was amplified by PCR and cloned into pEGFP-C1 expression vector containing green fluorescent protein. The eukaryotic expression vector pEGFP-C1-E2 was constructed and transfected into NP69 -SV40T cells. After transfection, Western blot, flow cytometry and CCK8 assay were performed to observe the effect on proliferation of NP69-SV40T cells. Results: The pEGFP-C1-E2 expression vector was successfully cloned and constructed. The expression of pEGFP-C1-E2 was confirmed in NP69-SV40T cells. CCK8 and flow cytometry were used to detect the cell cycle. The results showed that low- HPV E2 transfected NP69-SV40T cells can promote cell proliferation, while Western blot detection of the cell cycle CDK2 and CDK4 found in the three groups (transfected group, control group and blank group) expression was no significant difference. CONCLUSION: Eukaryotic expression plasmid pEGFP-C1-E2 expressing E2 can effectively transfect NP69-SV40T, and E2 gene expression can promote cell proliferation after transfection. These results provide a new idea and method for further study of the occurrence and development of recurrent respiratory papillomatosis.